It is very important to elucidate and analyze the function and structure of a protein, since it may be directly connected with the treatment of a disease or the creation of a new drug, for instance. Therefore, intensive studies have been made for synthesizing or producing various proteins by various methods, examining the structures thereof, and elucidating the mechanisms of action and the roles thereof in living organisms. It is now well known that the function of a protein is decided not only by the sequence of amino acids constituting the protein and the chain length but also by an orderly three-dimensional structure (higher order structure) it exhibits.
From the industrial viewpoint as well, the advancement of genetic engineering has made it possible to mass-produce various recombinant proteins for the use thereof in a wide range of industries, for example in drug manufacture, food processing and clinical diagnosis. Further, the development of the vector technology has made it possible for the technology of mass-producing target proteins in such microorganisms as Escherichia coli and yeasts to be practiced in a simple and easy manner using small amounts of resources and with good reproducibility.
However, many of the proteins expressed in recombinants are not orderly in three-dimensional structure or not controlled in higher order structure but often form inactive small-particle granules called inclusion bodies. Therefore, in production processes using Escherichia coli, a procedure is necessary for unfolding such inclusion bodies and converting them to soluble proteins having an orderly three-dimensional structure by higher order structure modification, namely for unfolding inclusion bodies and further refolding unfolded protein molecules.
This kind of refolding is applicable not only to proteins produced in Escherichia coli or yeasts but also to the regeneration of proteins inactivated by a certain cause, for example by thermal hysteresis; thus, it is a very important technology. Therefore, in the art, intensive studies have been made of this refolding technology and various methods have been proposed. However, the methods are mostly low in refolding rate; in addition, in many cases, favorable results were obtained incidentally with certain limited proteins (in particular specific low-molecular-weight proteins). Up to now, this refolding technology has not yet matured into a general and universal one which is applicable to various proteins, is efficient and economical, and gives high refolding rates.
Dialysis and dilution are old procedures used for refolding. The dialysis method comprises subjecting a protein to dialysis to refold the protein to its functional three-dimensional structure by gradually diluting a protein denaturing agent (unfolding agent; e.g. guanidine hydrochloride and/or urea) added in advance and substituting a buffer or the like therefor. Known as an example of application of this method is the stepwise dilution method which can raise the yield of refolding a protein to its functional three-dimensional structure by more slowly lowering the protein denaturing agent concentration (e.g. Non-Patent Document 1).
However, the dialysis method is not practical from the industrial point of view since the required volume of the dialyzing fluid generally amounts to at least 100 times the volume of the protein solution and, further, a period of several days is required.
The dilution method is widely used since it can be finished in a relatively short period of time and the volume required is relatively small, as compared with the dialysis method. The dilution method comprises excessively diluting, with a buffer or the like, a solution of a protein unfolded by addition of a protein denaturing agent (unfolding agent) to thereby refold the protein from its unfolded state to its functional three-dimensional structure. While the dilution method is a most simple and low-cost method of refolding a protein to its functional three-dimensional structure, the refolding yield rate is low (e.g. Non-Patent Document 2) and the high dilution ratio leads to a low yield under the existing circumstances.
An attempt has also been made to use an adsorptive separation column for refolding. When a protein or thioredoxin unfolded with urea/guanidine hydrochloride is subjected to gel filtration, refolding thereof occurs during gel filtration (Non-Patent Document 3). However, this method cannot always give sufficiently high refolding rates; generally, no satisfactory results can be obtained with other proteins. It has further been reported that when a protein solubilized with 8 M urea is adsorbed on a column with the molecular chaperone GroEL, a kind of protein promoting the refolding of a structurally denatured protein, immobilized thereon and then eluted with a solution containing 2 M potassium chloride and 2 M urea, refolding of the eluted protein occurs (Non-Patent Document 4). However, such refolding is observed only with a very limited number of proteins, for example cyclophilin A. In particular, under the present conditions, the use of a molecular chaperone, which is a certain kind of template, cannot serve at all when the protein to be refolded is incompatible with the template.
In some cases, a metal chelate is used as a substance to be immobilized on a column in lieu of the refolding promoting protein. When a His6tag-fused protein unfolded with an aqueous solution containing guanidine hydrochloride and urea is adsorbed on a resin with a nickel chelate immobilized thereon and the column is then washed with an unfolding agent-free buffer, refolding of the fused protein occurs (Non-Patent Document 5). However, the situation is the same: this method is applicable only to such protein and the resin preparation is complicated and results in increased costs.
There are also reports saying that when β-cyclodextrin or cycloamylose is used as an artificial chaperone and a protein unfolded with a surfactant is added to a solution of such chaperone, the surfactant is removed in the manner of inclusion in the artificial chaperone and, in this process, the protein is refolded (Non-Patent Document 6 to 8). However, such method is successfully applicable only to carbonic anhydrase B and the like. Furthermore, the method cannot be carried out repeatedly; hence, it is an expensive method.
In spite of such various proposed methods of refolding as discussed above, problems are still encountered in refolding unfolded proteins, namely low yields resulting from high-ratio dilution of the proteins as well as low purity levels.
Non-Patent Document 1: J. Biol. Chem. 2003 Mar. 14; 278(11):8979-8987
Non-Patent Document 2: J. Immunol. Methods. 1998 Oct. 1; 219(1-2):119-129
Non-Patent Document 3: Biochemistry, Vol. 26 (1987) 3135-3141
Non-Patent Document 4: Natl. Acad. Sci. USA, Vol. 94 (1997) 3576-3578
Non-Patent Document 5: Life Science News (Japan Ed.) Vol. 3 (2001) 6-7
Non-Patent Document 6: J. Am. Chem. Soc. Vol. 117 (1995) 2373-2374
Non-Patent Document 7: J. Biol. Chem. Vol. 271 (1996) 3478-3487
Non-Patent Document 8: FEBS Lett. Vol. 486 (2000) 131-135